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Administrative data

Description of key information

Skin irritation: Following exposure of the EpiDerm(TM) Model to the test item, pNMC oxide (8:1:1), the mean relative viability was 115.8% compared to the negative control value. This is above the threshold of 50%, therefore under the condition of this assay the test item was considered to be non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

Eye irritation: Severe corneal swelling change (mean = -16.5%) was observed during the four-hour observation period on three test item treated eyes. Severe cornea opacity change (severity 4) was observed on one test item treated eye. In the other two cases the cornea opacity change was not detected. Moderate fluorescein retention change (mean= 2.50) was noted on three test item treated eyes. The test item fully stuck on all cornea surfaces and the test item could not be removed during the study, despite best efforts to wash it off.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Remarks:
EpiDermTM Model (EPI-200-SIT)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 June 2020 - 28 July 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
(18 June 2019)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch/Lot number: PVX-144A PVX14
Description: Grey Powder
Purity: 100% (7.44% Co ; 6.99% Mn ; 59.67% Ni)
Manufacturer: Umicore
Expiry date: 30 April 2021
Storage conditions: Controlled room temperature (15-25 °C, ≤70% relative humidity). Protected from light and humidity (store in a tightly closed container).
The Certificate of Analysis is attached in Appendix 1 of the study report.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDerm™ Model (EPI-200-SIT), Source: MatTek, Bratislava, Slovakia, Lot No.:30874, Expiry Date: 19 June 2020
Justification for test system used:
The EpiDermTMhas been validated for irritation testing in an internation l validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD N° 439); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Remarks:
the test item was applied in the original form
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
EpiDerm™ Model (EPI-200-SIT) (Source: MatTek, Bratislava, Slovakia, Lot No.:30874, Expiry Date: 19 June 2020), The preparation of dead epithelium units involved the following procedure performed on living epithelium units (Manufacturer: MatTek, Bratislava, Slovak Republic., Batch No.: 30868).
- Production date:
Fresh = 17 June 2020; for freezing epidermal: 27 May 2020
- Date of initiation of testing:
17 June 2020

EpiDerm™ Model (EPI-200-SIT) (Source: MatTek, Bratislava, Slovakia, Lot No.:30874, Expiry Date: 19 June 2020) units consist of normal, human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis (0.6 cm2). It’s 3D structure consisting of organized and proliferative basal cells, spinous and granular layers, and cornified epidermal layers are mitotically and metabolically active. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.
The preparation of dead epithelium units involved the following procedure performed on living epithelium units (Manufacturer: MatTek, Bratislava, Slovak Republic., Batch No.: 30868). The process entailed placing the living epithelium units in a 24-well plate and the epidermis units were frozen (approximately -20 °C) overnight. The next day the killed tissues were thawed at room temperature approximately 1 hour and the units were frozen again. This method was performed two times, then the units were kept frozen until use (End of preparation: 28 May 2020, the frozen units are used up to 1 year after freezing). Further use of killed tissues was similar to living tissues.


TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
The plates with the test item, negative and positive control treated epidermis units were incubated for 35 minutes (± 1 minute) to the humidified incubator at 37°C with 5 % CO2, in a >95% humidified atmosphere and for the exposure time of 25 minutes (± 0.5 minute) at room temperature (24.6-27.1°C).
- Temperature of post-treatment incubation (if applicable):
day 0: After rinsing, the units were placed into the plate wells with fresh Assay Medium (0.9 mL/well) below them and then incubated for 24 hours (± 2hours) at 37°C in an incubator with 5 % CO2, in a >95 RH % humidified atmosphere.
day 1: At the end of the 24 hours incubation period the units were placed into the plate wells with fresh Assay Medium (0.9 mL/well) below them and then incubated for 18 hours (± 2hours) at 37°C in an incubator with 5 % CO2, in a >95 RH % humidified atmosphere

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
100 ml DPBS each, 20 tim. Additional rinsing was used (10 times) because the test item stuck the epidermal surface. No change was observed after the additional rinsing.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
300 µL MTT medium (1 mg MTT / ml medium)
- Incubation time: 3 hours
- Spectrophotometer:
Thermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14, Date of calibration: 13 August 2018, calibration is valid until August 2020
- Wavelength:
570 nm

NUMBER OF REPLICATE TISSUES:
3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues and killed tissues
- Procedure used to prepare the killed tissues (if applicable):
The process entailed placing the living epithelium units in a 24-well plate and the epidermis units were frozen (approximately -20 °C) overnight. The next day the killed tissues were thawed at room temperature approximately 1 hour and the units were frozen again. This method was performed two times, then the units were kept frozen until use
- N. of replicates : Two additional test item-treated living tissues were used for the non-specific optical density (OD) evaluation. Furthermore, two additional test item-treated killed epidermis and two negative control treated killed epidermis were used. To avoid a possible double correction for colour interference, for non-specific colour in killed tissues, two additional disks were used.
- Method of calculation used:
• Non-Specific MTT reduction calculation (NSMTT%): NSMTT% = [(ODKT- ODKNC) / ODNC] × 100
ODKNC: negative control killed tissues OD ODKT: test item treated killed tissues OD ODNC: negative control OD
If NSMTT % is ≤ 30%, then true MTT metabolic conversion (TODTT) is undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: test item treated viable tissues
• The % relative viability (RV%) for each test item replicate is calculated relative to the
mean negative control:
RV 1% = [TODTT1 / mean ODNC] × 100 RV 2% = [TODTT2 / mean ODNC] × 100 RV 3% = [TODTT3 / mean ODNC] × 100
• The mean value of the 3 individual relative viability % for test item is calculated: Mean Relative Viability % = (RV1% + RV2% + RV3%) / 3
If NSMTT% is > 30% relative to the negative control: additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.

PREDICTION MODEL / DECISION CRITERIA
The irritation potential of test items can be classified according to the United Nations Globally Harmonized System of Classification and Labelling of Chemicals, and a similar system is used in CLP. In the present study, the irritancy potential of test items is predicted by the mean tissue viability of tissues exposed to the test item. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean relative viability of three individual tissues after 60 minutes exposure to the test item and 42 hours post incubation is less or equal (≤) to 50% of the mean viability of the negative controls. In case the test chemical is found to be non-corrosive, and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%, the test chemical is considered to be irritant to skin in accordance with UN GHS Category 2. The test item may be considered to be non-irritant to skin in accordance with UN GHS (No Category), if the mean relative viability of three individual tissues after 60 minutes exposure to the test item and 42 hours post incubation is more than (>) 50% of the mean viability of the negative controls.

Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
approx. 47 mg
- Concentration (if solution):
applied in the original form on 25µl DPBS


NEGATIVE CONTROL
- Amount(s) applied (volume or weight):
30µl
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight):
30µl
- Concentration (if solution):

Additional control
Two additional test item treated viable tissues for colour control.

NSMTT and killed controls
30 μL of negative control (DPBS) or positive control (5% (w/v) SDS solution) or 30 μL of test item were added to each additional control skin unit by using a suitable pipette.
Duration of treatment / exposure:
The plates with the test item, negative and positive control treated epidermis units were incubated for 35 minutes (± 1 minute) to the humidified incubator at 37°C with 5 % CO2, in a >95% humidified atmosphere and for the exposure time of 25 minutes (± 0.5 minute) at room temperature (24.6-27.1°C).
Duration of post-treatment incubation (if applicable):
After rinsing, the units were placed into the plate wells with fresh Assay Medium (0.9 mL/well) below them and then incubated for 24 hours (± 2hours) at 37°C in an incubator with 5 % CO2, in a >95 RH % humidified atmosphere.
At the end of the 24 hours incubation period the units were placed into the plate wells with fresh Assay Medium (0.9 mL/well) below them and then incubated for 18 hours (± 2hours) at 37°C in an incubator with 5 % CO2, in a >95 RH % humidified atmosphere.
Number of replicates:
test item: 3 replicates
negative controls: 3 replicates
positive controls: 3 replicates
As the test item was coloured, two additional test item-treated living tissues were used for the non-specific optical density (OD) evaluation. Furthermore, two additional test item-treated killed epidermis and two negative control treated killed epidermis were used. To avoid a possible double correction for colour interference, for non-specific colour in killed tissues, two additional disks were used.
Irritation / corrosion parameter:
other: mean relative viability
Value:
115.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Additional controls

As the test item was coloured (grey), two additional test item-treated living tissues were used to determine the non-specific OD value and a non-specific colour percentage (NSCliving%). Results of this non-specific colour determination are shown in Table 1. The mean value for optical density (measured at 570 nm) was 0.011, and the NSCliving% value for the test item was calculated as 0.7%. This value was below 5%, therefore additional data calculation was not necessary.

Table 1: Optical Density (OD) and the Calculated Non Specific Colour % (NSCliving%) of the Additional Control Tissues

 Additional data        Optical Density (OD)  NCS% (living)
     Measured  Blank corrected  
 Treated with pNMCoxide (8:1:1)  1  0.052  0.006  
   2  0.062  0.016

 0.7

   mean  --  0.011  

Notes:
1. Mean blank value was 0.046.
2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Based on the physical, chemical properties of the test item, NSMTT control was used in this study. Therefore, additional controls and data calculations were necessary to exclude the false estimation of viability. Results of the additional controls on killed epidermis are shown in

Table 2. Based on these observed mean OD (0.000), the calculated NSMTT% was 0.00%.

Table 2: Optical Density (OD) and the calculated Non-Specific MTT Reduction (NSMTT) of the Additional Control Tissues (Killed Epidermis)

 Additional control

(killed epidermis)

 Optical Density (OD)        NSMTT  NSMTT%
     Measured  Blank Corrected    
 

 Treated with pNMCoxide (8:1:1)

 1

2

0.089

0.085 

0.043

0.039 

0.000

*-0.004 

 
   mean  --  0.041  0.000  0.0
  Treated with DPBS

1

2

0.087

0.091

0.041

0.045

   
    mean  -- 0.043    --

Notes:
1. Mean blank value was 0.046.
2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).
1. * Excluded (negative volume)

Two additional test item-treated killed tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.008, Non- Specific Colour % (NSCkilled%) was calculated as 0.5% (see Table 3).

Table 3: Optical Density (OD) and the calculated Non Specific Colour % (NSCkilled%) of the Additional Control Tissues (Killed Epidermis)

 Additional control (killed epidermis)        Optical Density (OD)

 NSC%

(killed)

     Measured  Blank Corrected  
  

Treated with pNMCoxide (8:1:1)

 1

2

0.051

0.057

0.005

0.011 

 0.5
   mean  --  0.008  

Notes:
1. Mean blank value was 0.046.
2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Viability Results

The results of the OD measured at 570 nm for the test item, positive and negative controls as well as the calculated relative viability percent values (% RV) are presented in Table 4. The OD values for the test item treated skin samples showed 115.8% relative viability compared to the negative control (after adjustment for non-specific MTT reduction).

Table 4: Optical Density (OD) and the Calculated Relative Viability % of the Samples

 Substance        Optical Density (OD)

 Viability

(%RV)

 Standard

Deviation (SD)

     Measured  Blank corrected    

 Negative control:

DPBS

1

2

1.720

1.717

1.673 

1.674

1.671

1.627 

101.0

100.8

98.1 

-- 

--

--

   mean  --  1.658  100.0  1.6

 Positive control:

5% (w/v) SDS solution

1

2

3

0.090

0.079

0.086 

0.044

0.033

0.040 

2.6

2.0

2.4 

--

--

-- 

   mean  --  0.039  2.3  0.3

 Test item: 

pNMCoxide (8:1:1)

1

2

1.860

2.041

1.996 

1.814

1.995

1.950 

109.4

120.4

117.7 

--

--

-- 

   mean  --  1.920  115.8  5.7

Notes:
1. Mean blank value was 0.046.
2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Interpretation of results:
GHS criteria not met
Remarks:
No indication of irritation
Conclusions:
Following exposure of the EpiDerm(TM) Model to the test item, pNMC oxide (8:1:1), the mean relative viability was 115.8% compared to the negative control value. This is above the threshold of 50%, therefore under the condition of this assay the test item was considered to be non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.
In conclusion, in this in vitro EpiDermTM model test with pNMC oxide (8:1:1) (Batch number: PVX-144A PVX14), the results indicate that the test item is non-irritant to skin.
Executive summary:

An in vitro skin irritation test of pNMC oxide (8:1:1) test item was performed in a reconstructed human epidermis model. EpiDerm(TM) Model is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue; CAS number: 298-93-1) assay. The irritation potential of the test item was evaluated according to the OECD No. 439 guideline.

Disks of EpiDerm(TM) Model (three units) were treated with the test item and incubated for 35 minutes 37 degrees Celcius, 5 %CO2 ,>95 RH% humidified atmosphere and 25 minutes at room temperature. Exposure of the test item was terminated by rinsing with Dulbecco’s Phosphate Buffered Saline (DPBS). The epidermis units were then incubated at 37 degrees Celcius for 24 hours in an incubator with 5% CO2, in a >95% humidified atmosphere and 37 degrees Celcius for 18 hours in an incubator with 5% CO2, in a >95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 degrees Celcius in an incubator with 5% CO2 protected from light, in a >95% humidified atmosphere. The precipitated formazan crystals were then extracted using extracting solution (MTT-100- EXT) for 2 hours with shaking at room temperature and quantified spectrophotometrically at 570 nm.

The negative control epidermis units were treated with DPBS, whilst the positive control epidermis units were treated with 5% (w/v) Sodium Dodecyl Sulphate (SDS) (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. The possible MTT interaction potential of the test item was examined using two additional test item treated and two negative control treated killed epidermis units. Furthermore, to avoid a possible double correction for colour interference, a third set of controls (two additional disks) for non-specific colour in killed tissues (NSCkilled) were used. For each treated tissue, the viability was expressed as a percentage relative to the negative control. If the mean relative viability is less orequal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

Following exposure of the EpiDerm(TM) Model to the test item, pNMC oxide (8:1:1), the mean relative viability was 115.8% compared to the negative control value. This is above the threshold of 50%, therefore under the condition of this assay the test item was considered to be non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EpiDerm(TM) Model test with pNMC oxide (8:1:1), the results indicate that the test item is non-irritant to skin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Remarks:
Isolated Chicken Eye Test (ICET)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
8 June 2020 - 17 July 2020 (draft report)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch/Lot number: PVX-144A PVX14
Description: Grey Powder
Purity: 100% (7.44% Co ; 6.99% Mn ; 59.67% Ni)
Manufacturer: Umicore
Expiry date: 30 April 2021
Storage conditions: Controlled room temperature (15-25 °C, ≤70% relative humidity). Protected from light and humidity (store in a tightly closed container).
The Certificate of Analysis is attached in Appendix 1 of the study report.
Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. (Hungary)
- Number of animals: 7
- Characteristics of donor animals (e.g. age, sex, weight): 7 weeks old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Heads were collected by a slaughter house technician and heads transported to Charles River Laboratories Hungary Kft. at ambient temperature at the earliest convenience.
After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at Charles River Laboratories Hungary Kft. and processed within 2 hours of collection.
- Time interval prior to initiating testing: within 2 hours
- indication of any existing defects or lesions in ocular tissue samples: The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e. fluorescein retention and corneal opacity scores ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit.
- Indication of any antibiotics used: no information

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: ~100,9 mg onto the entire surface of the cornea attempting to cover the cornea surface uniformly
- the test item was applied in its original form, no vehicle was used

VEHICLE
not uses

NEGATIVE CONTROL
- Amount applied: 30 mg powdered Imidalzole

POSITIVE CONTROL
- Amount applied: 30 µl physiological saline
Duration of treatment / exposure:
10 seconds exposure time
Observation period (in vivo):
ex vivo after exposure: 4 hours
Number of animals or in vitro replicates:
test item: 3 eyes
positive control: 3 eyes
negative control: 1 eye
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e. fluorescein retention and corneal opacity scores ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit.
The eyeball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.
Each eye was located in chamber identified by a unique number within the Test Facility.
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoid too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time. No significant corneal thickness changes (-1.6% was observed in one eye and 1.7 was observed in one eye) and no corneal thickness changes were observed in the other eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES
test item: 3 eyes
positive control: 3 eyes
negative control: 1 eye

NEGATIVE CONTROL USED
physiological saline

POSITIVE CONTROL USED
powdered Imidalzole

APPLICATION DOSE
After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position, while the test material was applied onto the centre of the cornea.
TEST MATERIAL
- Amount applied: ~100,9 mg onto the entire surface of the cornea attempting to cover the cornea surface uniformly
- the test item was applied in its original form, no vehicle was used
NEGATIVE CONTROL
- Amount applied: 30 mg powdered Imidalzole
POSITIVE CONTROL
- Amount applied: 30 µl physiological saline

EXPOSURE TIME
10 seconds

OBSERVATION PERIOD
The negative and positive control eyes and all test item treated eyes were evaluated pre-treatment (as described in Section 11.2. Baseline assessments) and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible.
- Additional gentle rinsing with 3x20 mL saline was performed (with two different rinsing method) at each time point when the test material or the positive control material remaining on the cornea was observed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: the slit lamp microscope was focused such that the physiological saline solution appeared as a visible, clear (sharp) image as it moved across the cornea surface
- Damage to epithelium based on fluorescein retention: The fluorescein retention determination the settings of the slit lamp microscope was the same as for opacity assessment, but the green light filter was used
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: the slit lamp microscope was focused such that the physiological saline solution appeared as a visible, clear (sharp) image as it moved across the cornea surface
- Macroscopic morphological damage to the surface: In the test item treated eyes: the cornea opacity was not detected in two cases because the test item fully stuck to the all cornea surfaces. Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared at 240 minutes after the post-treatment rinse.
In the positive control group: Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared at 240 minutes after the post-treatment rinse.
No other morphological effect was observed in the study

- Others (e.g, histopathology): At the end of the procedure, the corneas were carefully removed from the eyes and placed individually into labelled containers of preservative fluid (10% neutral buffered formalin, Manufacturer: lach:ner, Batch number: PP/2020/01168, Expiry date: 07 February 2021). The corneas are available for potential histopathology, stored at room temperature.

SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA: decision criteria as indicated in the TG
Irritation parameter:
percent corneal swelling
Remarks:
at up to 75 min
Value:
-16.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Remarks:
1.6%; ICE Class I
Positive controls validity:
valid
Remarks:
9.8%, ICE Calss II
Remarks on result:
other: ICE Class IV
Irritation parameter:
percent corneal swelling
Remarks:
at up to 240 min
Value:
-16.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
1.6%; ICE Class I
Positive controls validity:
valid
Remarks:
20.7%, ICE Class III
Remarks on result:
other: ICE Class IV
Irritation parameter:
cornea opacity score
Remarks:
mean maximum cornal opacity change
Value:
4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0, ICE Class I
Positive controls validity:
valid
Remarks:
4, ICE Class IV
Remarks on result:
other: ICE Class IV
Irritation parameter:
fluorescein retention score
Remarks:
mean fluorescein retention change
Value:
2.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0, ICE Cmass I
Positive controls validity:
valid
Remarks:
3, ICE Class IV
Remarks on result:
other: ICE Class III
Irritation parameter:
other:
Positive controls validity:
other: Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared at 240 minutes after the post-treatment rinse
Remarks on result:
other: Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared at 240 minutes after the post-treatment rinse.
Other effects / acceptance of results:
Other observations

Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared at 240 minutes after the post-treatment rinse.
Overall ICE Class: 1xIII 2xIV
Severe corneal swelling change was observed on three test item treated eyes. The opacity change was not detected because the test item fully stuck on all cornea surfaces and the test item could not be removed during the study, despite best efforts to wash it off. Based on this in vitro eye irritation study in isolated chicken eyes with Nickel Cobalt Manganese oxide (8:1:1), for OECD/GHS classification the test item was severely irritant. UN GHS Classification: Category 1. The results that were visible under the test item, which adhered to the cornea, all indicate a Category 1 result for the test item, although the number of observations were limited by the test item that could not be removed.

POSITIVE CONTROL
Other observations: Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared at 240 minutes after the
post-treatment rinse.
Overall ICE Class: 1xIII 2xIV
Based on these observations, the positive control substance Imidazole was classified as severe irritant according to the EU regulations. UN GHS Classification: Category 1.

NEGATIVE CONTROL
Other observations: none
Overall ICE Class: 3xI
The negative control Physiological saline was classified as non-irritating, UN GHS Classification: No Category.

The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical control data range. This study was considered to be valid.

The mean values of the treated eyes for maximum corneal thickness change, corneal opacity change and fluorescein retention change are given below.

TEST ITEM: pNMC-oxide (8:1:1)

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

-16.5%*

IV

Mean maximum corneal swelling at up to 240 min

-16.5 %

IV

Mean maximum corneal opacity change

4.00**

IV

Mean fluorescein retention change

2.50

III

Other Observations

Test item was stuck on all cornea surfacesafter the post-treatment rinse. The cornea surfaces were not cleared at 240 minutes after the post-treatment rinse.

Overall ICE Class

1xIII 2xIV

*The mean corneal swelling at 240 min was -16.5%. As no severe loosening of the epithelium and no other morphological effects were observed, the mean maximum corneal swelling is considered to be severe and therefore classified as ICE class: IV.

**Severe cornea opacity change (severity 4) was observed on one test item treated eye. In the other two cases the cornea opacity change was not detected we considered as 4.

Severe corneal swelling change was observed on three test item treated eyes.The opacity change was not detected because the test item fully stuck on all cornea surfaces and the test item could not be removed during the study, despite best efforts to wash it off. Based on thisin vitroeye irritation study in isolated chicken eyes withNickel Cobalt Manganese oxide (8:1:1), for OECD/GHS classificationthe test item was severely irritant.UN GHS Classification: Category 1.The results that were visible under the test item, which adhered to the cornea, all indicate a Category 1 result for the test item, although the number of observations were limited by the test item that could not be removed.

POSITIVE CONTROL

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

9.8%

II

Mean maximum corneal swelling at up to 240 min

20.7%

III

Mean maximum corneal opacity change

4.00

IV

Mean fluorescein retention change

3.00

IV

Other Observations

Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared at 240 minutes after the
post-treatment rinse.

Overall ICE Class

1xIII 2xIV

Based on these observations, the positive control substance Imidazole was classified as severe irritant according to the EU regulations. UN GHS Classification: Category 1.

NEGATIVE CONTROL

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

1.6%

I

Mean maximum corneal swelling at up to 240 min

1.6%

I

Mean maximum corneal opacity change

0.00

I

Mean fluorescein retention change

0.00

I

Other Observations

None

Overall ICE Class

3xI

 The negative control Physiological saline was classified as non-irritating, UN GHS Classification: No Category.

VALIDITY CRITERIA

The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical control data range. This study was considered to be valid.

SUMMARY TABLE FOR UN GHS CLASSIFICATION

Criteria for “No category” (all true)

 

3 endpoints classed as I or 2 endpoints classed as I and 1 endpoint classed as II or 1 endpoint classed as I and 2 endpoints classed as II:

False

No severe corneal morphological changes:

True

Test item was not stuck to the cornea at 240 minutes after the post-treatment rinse:

False

 

Criteria for “Category 1” (one or more true)

 

2 or more endpoints classed as IV:

True

Corneal opacity ≥ 3 at 30 min (in at least 2 eyes):

False

Corneal opacity = 4 at any time point (in at least 2 eyes):

False

Severe loosening of epithelium (in at least 1 eye):

False

 

Criteria for “No prediction can be made” (one or two true)

 

Based on the endpoints not classifiable for No Category, or for Category 1:

False

Particles of test item were stuck to the cornea and could not be washed off during the study:

True

Interpretation of results:
study cannot be used for classification
Conclusions:
Observed effects in the in vitro OECD438 test indicate that pNMC oxide (8:1:1) should be classified as Eye Dam. Cat.1. Severe corneal swelling change (mean = -16.5%) was observed during the four-hour observation period on three test item treated eyes. Severe cornea opacity change (severity 4) was observed on one test item treated eye. In the other two cases the cornea opacity change was not detected. Moderate fluorescein retention change (mean= 2.50) was noted on three test item treated eyes. However, the test conditions did not meet those that are specified in the testing procedure: the test item fully stuck on all cornea surfaces and the test item could not be removed during the study, despite best efforts to wash it off.
Based on the test data it is not possible to draw a conclusive decision on the potential hazard of pNMC oxide (8:1:1) for the eye irritation endpoint. Therefore the no-classification for the eye – as determined theoretically with MeClas - is provisionally assigned to this substance.
Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD
No. 438 guideline (25 June 2018).

After the zero reference measurements, the eyes were held in a horizontal position and the test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered in all cases. After 10 seconds exposure time, the surface of the eyes was rinsed with physiological saline solution. Three eyes were treated with approximately 100.9 mg test item. Three positive control eyes were treated in a similar way with approximately100.9 mg powdered Imidazole and the negative control eye was treated with 30 µL of physiological saline (0.9% (w/v) NaCl solution). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in experiment. Thus, the study was considered to be valid.

Severe corneal swelling change (mean = -16.5%) was observed during the four-hour observation period on three test item treated eyes. Severe cornea opacity change (severity 4) was observed on one test item treated eye. In the other two cases the cornea opacity change was not detected. Moderate fluorescein retention change (mean= 2.50) was noted on three test item treated eyes. The test item fully stuck on all cornea surfaces and the test item could not be removed during the study, despite best efforts to wash it off.

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The results of the tests with pNMC oxide show that the substance is non-irritant to skin.

Observed effects in the in vitro OECD438 test indicate that pNMC oxide should be classified as Eye Dam. Cat.1, but the test conditions did not meet those that are specified in the testing procedure: it was not possible to remove pNMC oxide (by rinsing) after the initial exposure.

It is not possible to draw a conclusive decision on the potential hazard of pNMC oxide (8:1:1) that is based on test data. Therefore the Eye Irrit. Cat.2 classification that is determined theoretically with MeClas is assigned to this substance.

 

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